title: Directed evolution of polymerases to accept nucleotides with nonstandard hydrogen bond patterns.
abstract: Artificial genetic systems have been developed by synthetic biologists over the past two decades to include additional nucleotides that form additional nucleobase pairs independent of the standard T:A and C:G pairs. Their use in various tools to detect and analyze DNA and RNA requires polymerases that synthesize duplex DNA containing unnatural base pairs. This is especially true for nested polymerase chain reaction (PCR), which has been shown to dramatically lower noise in multiplexed nested PCR if nonstandard nucleotides are used in their external primers. We report here the results of a directed evolution experiment seeking variants of Taq DNA polymerase that can support the nested PCR amplification with external primers containing two particular nonstandard nucleotides, 2-amino-8-(1'-β-d-2'-deoxyribofuranosyl)imidazo[1,2-a]-1,3,5-triazin-4(8H)-one (trivially called P) that pairs with 6-amino-5-nitro-3-(1'-β-d-2'-deoxyribofuranosyl)-2(1H)-pyridone (trivially called Z). Variants emerging from the directed evolution experiments were shown to pause less when challenged in vitro to incorporate dZTP opposite P in a template. Interestingly, several sites involved in the adaptation of Taq polymerases in the laboratory were also found to have displayed "heterotachy" (different rates of change) in their natural history, suggesting that these sites were involved in an adaptive change in natural polymerase evolution. Also remarkably, the polymerases evolved to be less able to incorporate dPTP opposite Z in the template, something that was not selected. In addition to being useful in certain assay architectures, this result underscores the general rule in directed evolution that "you get what you select for".
authors.affiliation: Foundation for Applied Molecular Evolution and The Westheimer Institute of Science and Technology, Gainesville, Florida 32601, United States.
authors.forename: Roberto
authors.initials: R
authors.lastname: Laos
authors.forename: Ryan
authors.initials: R
authors.lastname: Shaw
authors.forename: Nicole A
authors.initials: NA
authors.lastname: Leal
authors.forename: Eric
authors.initials: E
authors.lastname: Gaucher
authors.forename: Steven
authors.initials: S
authors.lastname: Benner
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D019020:Directed Molecular Evolution; D019143:Evolution, Molecular; D006859:Hydrogen / Q000378:metabolism; D006860:Hydrogen Bonding; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism*; D019914:Taq Polymerase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013824:Thermus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D009711:Nucleotides; D006859:Hydrogen; D019914:Taq Polymerase